Studies On The Screening And Antimicrobial Mechanism Of Biological Pesticide Derived From Marine Bacteria

A series of agricultural dieases and disasters caused by agricultural pathogens seriously threaten food production safety and cause great economic loss in China.Microbial source biological pesticides have attracted more and more attention due to its low toxicity and environmental friendliness.As the largest ecosystems in the word,marine environment has a variety of microbial strains and active material resources.Marine environment is rich in the resources of active compound candidates and provides novel biological pesticide precursors.In our research,the agricultural pathogenic microorganisms,including Vibrio anguillarum,Phytophthora nicotianae and Magnaporthe grisea,were used as the research object to obtain the antagonistic bacteria and their active products.Furthermore,the antimicrobial mechanisms of obtained active compounds were also explored with proteomic and molecular biology techniques.Phenazine and its derivatives are very important secondary metabolites produced from Pseudomonas spp.and have exhibited broad-spectrum antifungal and antibacterial activities.However,till date,there are few reports about marine derived Pseudomonas and its production of phenazine metabolites.In this study,we isolated a marine strain Pseudomonas aeruginosa PA31 x which produced natural product inhibiting the growth of V.anguillarum,one of the most serious bacterial pathogens in marine aquaculture.Combining high-resolution electro-spray-ionization mass spectroscopy and nuclear magnetic resonance spectroscopy analyses,the functional compound against V.anguillarum C312 was demonstrated to be phenazine-1-carboxylic acid(PCA),an important phenazine derivative.Molecular studies indicated that the production of PCA by P.aeruginosa PA31 x was determined by gene clusters phz1 and phz2 in its genome.Electron microscopic results showed that treatment of V.anguillarum C312 with PCA led complete lysis of bacterial cells with fragmented cytoplasm being released to the surrounding environment.Additional evidence indicated that reactive oxygen species generation preceded PCA-induced microbe and cancer cell death.Notably,treatment with PCA gave highly significant protective activities against the development of V.anguillarum C312 on zebrafish.Additionally,the marine derived PCA was further found to effectively inhibit the growth of agricultural pathogens P.nicotianae.Taken together,this study reveals that marine Pseudomonas derived PCA carries antagonistic activities against both aquacultural and agricultural pathogens,which broadens the application fields of PCA.Rice blast caused by phytopathogen M.grisea posesses a serious threat to the global food security and is difficult to control.Bacillus species have been extensively explored for the biological control of many fungal diseases.In the present study,marine bacterium Bacillus subtilis BS155 showed a strong antifungal activity against M.grisea.The active metabolites were isolated and identified as cyclic lipopeptides(CLPs)of the fengycin family,named fengycin BS155,by the combination of high-performance liquid chromatography(HPLC),electrospray ionisation mass spectrometry(ESI-MS)and tandem mass spectrometry(ESI-MS/MS).Analyses using scanning and transmission electron microscopy revealed that fengycin BS155 caused morphological changes in the plasma membrane and cell wall of M.grisea hyphae.Using comparative proteomic and biochemical assays,fengycin BS155 was demonstrated to reduce the mitochondrial membrane potential(MMP),induce bursts of reactive oxygen species(ROS)and down-regulate the expression level of ROS scavenging enzymes.Simultaneously,fengycin BS155 caused chromatin condensation in fungal hyphal cells,which led to the up-regulation of DNA repair-related proteins expression and the cleavage of poly(ADP-ribose)polymerase(PARP).Taken together,our results indicate that fengycin BS155 acts by inducing membrane damage and dysfunction of organelles,disruption of MMP,oxidative stress,and chromatin condensation,resulting in M.grisea hyphal cell death.Therefore,fengycin BS155 and its parent bacterium are very promising candidates for the biological control of M.grisea and the associated rice blast.